RESUMO
A series of arylketo-containing P1-P3 linked macrocyclic BACE-1 inhibitors were designed, synthesized, and compared with compounds with a previously known and extensively studied corresponding P2 isophthalamide moiety with the aim to improve on permeability whilst retaining the enzyme- and cell-based activities. Several inhibitors displayed substantial increases in Caco-2 cell-based permeability compared to earlier synthesized inhibitors and notably also with retained activities, showing that this approach might yield BACE-1 inhibitors with improved properties.
RESUMO
Novel BACE-1 inhibitors with a hydroxyethylene central core have been developed. Modified P1´ and extended P1 substituents were incorporated with the aim to explore potential interactions with the S1´ and the S1-S3 pocket, respectively, of BACE-1. Inhibitors were identified displaying IC50 values in the nanomolar range, i.e. 69 nM for the most potent compound. Possible inhibitor interactions with the enzyme are also discussed.
RESUMO
A series of P1-P3 linked macrocyclic BACE-1 inhibitors containing a hydroxyethylamine (HEA) isostere scaffold has been synthesized. All inhibitors comprise a toluene or N-phenylmethanesulfonamide P2 moiety. Excellent BACE-1 potencies, both in enzymatic and cell-based assays, were observed in this series of target compounds, with the best candidates displaying cell-based IC(50) values in the low nanomolar range. As an attempt to improve potency, a phenyl substituent aiming at the S3 subpocket was introduced in the macrocyclic ring. X-ray analyzes were performed on selected compounds, and enzyme-inhibitor interactions are discussed.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Etilaminas/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/química , Etilaminas/síntese química , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
We herein describe the design and synthesis of a series of BACE-1 inhibitors incorporating a P1-substituted hydroxylethylene transition state isostere. The synthetic route starting from commercially available carbohydrates yielded a pivotal lactone intermediate with excellent stereochemical control which subsequently could be diversified at the P1-position. The final inhibitors were optimized using three different amines to provide the residues in the P2'-P3' position and three different acids affording the residues in the P2-P3 position. In addition we report on the stereochemical preference of the P1'-methyl substituent in the synthesized inhibitors. All inhibitors were evaluated in an in vitro BACE-1 assay where the most potent inhibitor, 34-(R), exhibited a BACE-1 IC(50) value of 3.1 nM.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Etilenos/química , Linhagem Celular , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Several BACE-1 inhibitors with low nanomolar level activities, encompassing a statine-based core structure with phenyloxymethyl- and benzyloxymethyl residues in the P1 position, are presented. The novel P1 modification introduced to allow the facile exploration of the S1 binding pocket of BACE-1, delivered highly promising inhibitors.
Assuntos
Aminoácidos/química , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Desenho de Fármacos , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Cristalografia por Raios X , Humanos , Modelos Moleculares , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Inibidores de Proteases/química , Relação Estrutura-AtividadeRESUMO
Several highly potent novel HCV NS3 protease inhibitors have been developed from two inhibitor series containing either a P2 trisubstituted macrocyclic cyclopentane- or a P2 cyclopentene dicarboxylic acid moiety as surrogates for the widely used N-acyl-(4R)-hydroxyproline in the P2 position. These inhibitors were optimized for anti HCV activities through examination of different ring sizes in the macrocyclic systems and further by exploring the effect of P4 substituent removal on potency. The target molecules were synthesized from readily available starting materials, furnishing the inhibitor compounds in good overall yields. It was found that the 14-membered ring system was the most potent in these two series and that the corresponding 13-, 15-, and 16-membered macrocyclic rings delivered less potent inhibitors. Moreover, the corresponding P1 acylsulfonamides had superior potencies over the corresponding P1 carboxylic acids. It is noteworthy that it has been possible to develop highly potent HCV protease inhibitors that altogether lack the P4 substituent. Thus the most potent inhibitor described in this work, inhibitor 20, displays a K(i) value of 0.41 nM and an EC(50) value of 9 nM in the subgenomic HCV replicon cell model on genotype 1b. To the best of our knowledge this is the first example described in the literature of a HCV protease inhibitor displaying high potency in the replicon assay and lacking the P4 substituent, a finding which should facilitate the development of orally active small molecule inhibitors against the HCV protease.
Assuntos
Ciclopentanos/farmacologia , Inibidores Enzimáticos/farmacologia , Compostos Macrocíclicos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Ciclização , Ciclopentanos/síntese química , Ciclopentanos/química , Ácidos Dicarboxílicos/química , Relação Dose-Resposta a Droga , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Hepacivirus/enzimologia , Humanos , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/química , Modelos Moleculares , Conformação Molecular , Estereoisomerismo , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
Potent tetrapeptidic inhibitors of the HCV NS3 protease have been developed incorporating 4-hydroxy-cyclopent-2-ene-1,2-dicarboxylic acid as a new N-acyl-l-hydroxyproline mimic. The hydroxycyclopentene template was synthesized in eight steps from commercially available (syn)-tetrahydrophthalic anhydride. Three different amino acids were explored in the P1-position and in the P2-position the hydroxyl group of the cyclopentene template was substituted with 7-methoxy-2-phenyl-quinolin-4-ol. The P3/P4-positions were then optimized from a set of six amino acid derivatives. All inhibitors were evaluated in an in vitro assay using the full-length NS3 protease. Several potent inhibitors were identified, the most promising exhibiting a K(i) value of 1.1nM.
Assuntos
Ciclopentanos/síntese química , Ciclopentanos/farmacologia , Ácidos Dicarboxílicos/síntese química , Ácidos Dicarboxílicos/farmacologia , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Indicadores e Reagentes , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The HCV NS3 protease is essential for replication of the hepatitis C virus (HCV) and therefore constitutes a promising new drug target for anti-HCV therapy. Several potent and promising HCV NS3 protease inhibitors, some of which display low nanomolar activities, were identified from a series of novel inhibitors incorporating a trisubstituted cyclopentane dicarboxylic acid moiety as a surrogate for the widely used N-acyl-(4R)-hydroxyproline in the P2 position.
Assuntos
Antivirais/farmacologia , Ciclopentanos/farmacologia , Hepacivirus/efeitos dos fármacos , Inibidores de Serina Proteinase/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/síntese química , Antivirais/química , Cristalografia por Raios X , Ciclopentanos/síntese química , Ciclopentanos/química , Relação Dose-Resposta a Droga , Hepacivirus/enzimologia , Modelos Moleculares , Conformação Molecular , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
New and potent inhibitors of the malarial aspartic proteases plasmepsin (Plm) I and II, from the deadliest malaria parasite Plasmodium falciparum, have been synthesized utilizing Suzuki coupling reactions on previously synthesized bromobenzyloxy-substituted statine-like inhibitors. The enzyme inhibition activity has been improved up to eight times by identifying P1 substituents that effectively bind to the continuous S1-S3 crevice of Plasmepsin I and II. By replacement of the bromo atom in the P1 p-bromobenzyloxy-substituted inhibitors with different aryl substituents, several inhibitors exhibiting K(i) values in the low nanomolar range for both Plm I and II have been identified. Some of these inhibitors are also effective in attenuating parasite growth in red blood cells, with the best inhibitors, compounds 2 and 4, displaying 70% and 83% inhibition, respectively, at a concentration of 5 microM. The design was partially guided by the X-ray crystal structure disclosed herein of the previously synthesized inhibitor 1 in complex with plasmepsin II.
Assuntos
Antiprotozoários/síntese química , Antiprotozoários/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/química , Plasmodium falciparum/enzimologia , Animais , Antiprotozoários/química , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários , Relação Estrutura-AtividadeRESUMO
HIV-1 protease is a pivotal enzyme in the later stages of the viral life cycle which is responsible for the processing and maturation of the virus particle into an infectious virion. As such, HIV-1 protease has become an important target for the treatment of AIDS, and efficient drugs have been developed. However, negative side effects and fast emerging resistance to the current drugs have necessitated the development of novel chemical entities in order to exploit different pharmacokinetic properties as well as new interaction patterns. We have used X-ray crystallography to decipher the structure-activity relationship of fluoro-substitution as a strategy to improve the antiviral activity and the protease inhibition of C2-symmetric diol-based inhibitors. In total we present six protease-inhibitor complexes at 1.8-2.3 A resolution, which have been structurally characterized with respect to their antiviral and inhibitory activities, in order to evaluate the effects of different fluoro-substitutions. These C2-symmetric inhibitors comprise mono- and difluoro-substituted benzyloxy side groups in P1/P1' and indanoleamine side groups in P2/P2'. The ortho- and meta-fluorinated P1/P1'-benzyloxy side groups proved to have the most cytopathogenic effects compared with the nonsubstituted analog and related C2-symmetric diol-based inhibitors. The different fluoro-substitutions are well accommodated in the protease S1/S1' subsites, as observed by an increase in favorable Van der Waals contacts and surface area buried by the inhibitors. These data will be used in the development of potent inhibitors with different pharmacokinetic profiles towards resistant protease mutants.
Assuntos
Derivados de Benzeno/química , Derivados de Benzeno/farmacologia , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , HIV-1/enzimologia , Hidrocarbonetos Fluorados/química , Hidrocarbonetos Fluorados/farmacologia , Aminoácidos/química , Aminoácidos/metabolismo , Derivados de Benzeno/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Protease de HIV/química , Protease de HIV/genética , Protease de HIV/metabolismo , Inibidores da Protease de HIV/metabolismo , Humanos , Hidrocarbonetos Fluorados/metabolismo , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Ligação Proteica , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
Picomolar to low nanomolar inhibitors of the two aspartic proteases plasmepsin (Plm) I and II, from the malaria parasite Plasmodium falciparum, have been identified from sets of libraries containing novel statine-like templates modified at the amino and carboxy terminus. The syntheses of the novel statine templates were carried out in solution phase using efficient synthetic routes and resulting in excellent stereochemical control. The most promising statine template was attached to solid support and diversified by use of parallel synthesis. The products were evaluated for their Plm I and II inhibitory activity as well as their selectivity over cathepsin D. Selected inhibitors were, in addition, evaluated for their inhibition of parasite growth in cultured infected human red blood cells. The most potent inhibitor in this report, compound 16, displays Ki values of 0.5 and 2.2 nM for Plm I and II, respectively. Inhibitor 16 is also effective in attenuating parasite growth in red blood cells showing 51% inhibition at a concentration of 5 microM. Several inhibitors have been identified that exhibit Ki values between 0.5 and 74 nM for both Plm I and II. Some of these inhibitors also show excellent selectivity vs cathepsin D.
Assuntos
Acrilonitrila/síntese química , Antimaláricos/síntese química , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Dipeptídeos/síntese química , Tiofenos/síntese química , Acrilonitrila/análogos & derivados , Acrilonitrila/química , Acrilonitrila/farmacologia , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Ácido Aspártico Endopeptidases/química , Catepsina D/antagonistas & inibidores , Catepsina D/química , Técnicas de Química Combinatória , Dipeptídeos/química , Dipeptídeos/farmacologia , Humanos , Modelos Moleculares , Mimetismo Molecular , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários , Estereoisomerismo , Relação Estrutura-Atividade , Tiofenos/química , Tiofenos/farmacologiaRESUMO
A 4-amino-2-carboxymethyl-3-morpholinone structural motif derived from malic acid has been used to mimic d-Phe-Pro in the thrombin inhibiting tripeptide d-Phe-Pro-Arg. The arginine in D-Phe-Pro-Arg was replaced by the more rigid P1 truncated p-amidinobenzylamine (Pab). These new thrombin inhibitors were used to probe the inhibitor binding site of alpha-thrombin. The best candidate in this series of thrombin inhibitors exhibits an in vitro IC50 of 0.130 microM. Interestingly, the stereochemistry of the 4-amino-2-carboxymethyl-3-morpholinone motif is reversed for the most active compounds compared to that of a previously reported 2-carboxymethyl-3-morpholinone series. The X-ray crystal structure of the lead inhibitor cocrystallized with alpha-thrombin is discussed.
Assuntos
Morfolinas/química , Morfolinas/farmacologia , Trombina/antagonistas & inibidores , Trombina/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Morfolinas/síntese química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
New inhibitors of plasmepsin I and II, the aspartic proteases of the malaria parasite Plasmodium falciparum, are described. From paralell solution phase chemistry, several reversed-statine type isostere inhibitors, many of which are aza-peptides, have been prepared. The synthetic strategy delivers the target compounds in good to high overall yields and with excellent stereochemical control throughout the developed route. The final products were tested for their plasmepsin I and II inhibiting properties and were found to exhibit modest but promising activity. The best inhibitor exhibits K(i) values of 250 nM and 1.4 microM for Plm I and II, respectively.
Assuntos
Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Plasmodium falciparum/enzimologia , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Animais , Malária/tratamento farmacológico , Estrutura Molecular , Inibidores de Proteases/química , Proteínas de ProtozoáriosRESUMO
With the aim to develop inhibitors of the plasmepsin I and II aspartic proteases of the malaria parasite Plasmodium falciparum, we have synthesized sets of libraries from novel reversed-statine isosteres, using a combination of solution phase and solid phase chemistry. The synthetic strategy furnishes the library compounds in good to high overall yields and with excellent stereochemical control throughout the developed route. The products were evaluated for their plasmepsin I and II inhibiting properties and were found to exhibit modest but promising activity. The best inhibitor exhibits an in vitro activity of 28% inhibition of plasmepsin II at an inhibitor concentration of 0.5 microM (K(i) for Plm II=5.4 microM).
Assuntos
Aminoácidos/síntese química , Aminoácidos/farmacologia , Antimaláricos/síntese química , Antimaláricos/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Biblioteca de Peptídeos , Plasmodium/enzimologia , Animais , Reagentes de Ligações Cruzadas , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Proteínas de Protozoários , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The thrombin inhibitory tripeptide d-Phe-Pro-Arg has been mimicked using either cyclopentenedicarboxylic derivatives or a cyclohexenedicarboxylic derivative as surrogate for the P2 proline. In the P3 position, tertiary amides were optimized as d-Phe P3 replacements. The P1 arginine was, in all compounds, substituted with the more rigid and biocompatible 4-aminomethylbenzamidine. One of the novel inhibitors was cocrystallized with alpha-thrombin and subjected to X-ray analysis. From analysis of the X-ray crystal structure, new ligands were designed leading to significantly improved binding affinity, the lead candidate exhibiting an in vitro IC(50) of 49 nM.
Assuntos
Amidas/síntese química , Benzamidas/síntese química , Cicloexanos/síntese química , Ciclopentanos/síntese química , Ácidos Dicarboxílicos/síntese química , Inibidores de Serina Proteinase/síntese química , Trombina/antagonistas & inibidores , Amidas/química , Benzamidas/química , Cristalografia por Raios X , Cicloexanos/química , Ciclopentanos/química , Ácidos Dicarboxílicos/química , Ligantes , Modelos Moleculares , Mimetismo Molecular , Oligopeptídeos/química , Inibidores de Serina Proteinase/química , Relação Estrutura-AtividadeRESUMO
A morpholinone structural motif derived from D(+)- and L(-)-malic acid has been used as a mimic of D-Phe-Pro in the thrombin inhibiting tripeptide D-Phe-Pro-Arg. In place of Arg the more rigid P1 truncated p-amidinobenzylamine (Pab) or 2-amino-5-aminomethyl-3-methyl-pyridine have been utilized. The synthetic strategy developed readily delivers these novel thrombin inhibitors used to probe the alpha-thrombin inhibitor binding site. The best candidate in this series of thrombin inhibitors exhibits an in vitro IC(50) of 720 nM. The X-ray crystal structure of this candidate co-crystallized with alpha-thrombin is discussed.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Mimetismo Molecular , Morfolinas/química , Oligopeptídeos/química , Trombina/antagonistas & inibidores , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Modelos Moleculares , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
With the objective to prepare novel non-peptidic thrombin inhibitors, bioisosteres of the inhibitory tripeptide D-Phe-Pro-Arg chain have been examined. Thus, the P1 Arg was replaced with p-amidinobenzylamine, an elongated homologue of the same and with 2,5-dichloro benzylamine. The P2-P3, D-Phe-Pro, was replaced with a novel tartaric acid template coupled to a series of readily available, mainly lipophilic, amines. Some of these compounds exhibit promising thrombin inhibition activity in vitro, IC(50 ) approximately 5.9 microM.
Assuntos
Prolina/química , Inibidores de Serina Proteinase/síntese química , Tartaratos/química , Trombina/antagonistas & inibidores , Aminas/síntese química , Aminas/química , Aminas/farmacologia , Anticoagulantes/síntese química , Anticoagulantes/química , Anticoagulantes/farmacologia , Cristalografia por Raios X , Humanos , Concentração Inibidora 50 , Mimetismo Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-AtividadeRESUMO
The synthesis of the enantiomerically pure bis(hydroxymethyl)-branched cyclohexenyl and cyclohexyl purines is described. Racemic trans-4,5-bis(methoxycarbonyl)cyclohexene [(+/-)-6] was reduced with lithium aluminum hydride to give the racemic diol (+/-)-7. Resolution of (+/-)-7 via a transesterification process using lipase from Pseudomonas sp. (SAM-II) gave both diols in enantiomerically pure form. The enantiomerically pure diol (S,S)-7was benzoylated and epoxidized to give the epoxide 9. Treatment of the epoxide 9 with trimethylsilyl trifluoromethanesulfonate and 1,5-diazabicyclo[5.4.0]undec-5-ene followed by dilute hydrochloric acid gave (1R,4S,5R)-4,5-bis[(benzoyloxy)methyl]-1-hydroxycyclohex-2-ene (10). Acetylation of 10 gave (1R,4S,5R)-1-acetoxy-4,5-bis[(benzoyloxy)methyl]cyclohex-2-ene (11). (1R,4S,5R)-1-Acetoxy-4,5-bis[(benzoyloxy)methyl]cyclohex-2-ene (11) was converted to the adenine derivative 12 and guanine derivative 13 via palladium(0)-catalyzed coupling with adenine and 2-amino-6-chloropurine, respectively. Hydrogenation of 12 and 13 gave the correspondning saturated adenine derivative 14 and guanine derivative 15. (1R,4S,5R)-4,5-Bis[(benzoyloxy)methyl]-1-hydroxycyclohex-2-ene (10) was converted to the adenine derivative 16 and guanine derivative 17 via coupling with 6-chloropurine and 2-amino-6-chloropurine, respectively, using a modified Mitsunobu procedure. Hydrogenation of 16 and 17 gave the corresponding saturated adenine derivative 18 and guanine derivative 19. Compounds 12-19 were evaluated for activity against human immunodeficiency virus (HIV), but were found to be inactive. Further biological testings are underway.
RESUMO
The synthesis of 1,3-dioxolan-2-ylnucleosides and related chemistry is described. We have shown that 2-methoxy-1,3-dioxolane (6) reacts with silylated thymine and trimethylsilyl triflate to give the acyclic formate ester 1-[2-(formyloxy)ethyl]thymine (8) rather than 1-(1,3-dioxolan-2-yl)thymine (7). A tentative mechanism which could explain this result is discussed. On the other hand, 2-methoxy-1,3-dioxolane 13c reacts with silylated bases to give [4,5-bis(hydroxymethyl)-1,3-dioxolan-2-yl]nucleosides, thus representing the first examples of this novel class of compounds. The nature of the nucleobase and the hydroxyl protecting groups was found to have great influence on the reaction and on the stability of the nucleosides. Compounds 16 and 18 were found to be inactive when tested for anti HIV-1 activity in vitro.
RESUMO
The synthesis of [4,5-bis(hydroxymethyl)-1,3-oxathiolan-2-yl]nucleosides is described. 2,3-Epoxy alcohol 10 was converted in one pot into thioacetate 11. Treatment of 11 under mild alkaline conditions gave thiirane 12 with inversion of configuration at C-2. We also found that thioacetate 11 rearranges into thiirane 14 under mild acidic conditions. This rearrangement reaction was shown by independent synthesis to proceed with net retention of configuration at C-2. We have proposed a tentative mechanism which may explain the results obtained. Opening of thiiranes 12 and 14 followed by deprotection gave (2R,3R)-2-thiothreitol (23) and (2S,3R)-2-thioerythritol (25), respectively. Regioselective silylation of the primary hydroxyl groups of 23 followed by treatment with trimethyl orthoformate gave 2-methoxy-1,3-oxathiolanes 26 and 27. Condensation with silylated bases followed by deprotection and separation of the anomers gave the oxathiolanylnucleosides. Compounds 29-31, 34, and 35 were found to be inactive when tested for inhibition of HIV-1 activity in vitro.